Glucokinase is a key regulator of glucose homeostasis in the human body. Dysfunction in the regulation and/or activity of glucokinase causes a diverse range of diseases including maturity onset diabetes of the young (MODY) and hyperinsulinemia of infancy (HI). Human glucokinase is a monomeric enzyme that is allosterically regulated by its sugar substrate, D- glucose. This kinetic cooperativity is critical to maintaining proper glucose serum levels in vivo. The goal of this study is to elucidate the mechanistic basis for cooperativity in human glucokinase. The experiments described herein combine a powerful genetic selection system for human glucokinase activity with mechanistic enzymology to provide a kinetic and molecular description of the conformational transitions that give rise to cooperativity. The specific aims are: (1) To determine the mechanism of activation of hyperinsulinemia-linked glk mutations; (2) To investigate conformational heterogeneity in unliganded glucokinase via chemical quench-flow techniques; (3) To investigate the role of a secondary structural allosteric switch in glucokinase cooperativity; (4) To determine whether prolyl cis/trans isomerization is responsible for the slow interconversion of enzyme conformations. Achieving the objectives outlined in this application promises to impact the development of future glucokinase-targeted therapeutics for diabetes and hyperinsulinemia.